The control of protein levels in the cell was once thought to be almost entirely due to changes in transcription and translation. Today we understand that a significant part of this control occurs by regulating protein stability through the ubiquitin-proteasome system. This is particularly clear in the cell cycle, where transitions from one state to another are mediated by rapid changes in protein stability, but the general importance of post-translational modifications that affect protein stability is turning out to be quite common. In this grant, we propose to use protein microarrays to develop a general method for measuring post-translational modifications quantitatively, initially focusing on ubiquitination and sumoylation. The key feature of this approach is the use of concentrated cell extracts that are stably arrested in specific states of the cell cycle and that can perform cell cycle phase-appropriate reactions. This should provide a new approach to understanding regulation via post-translational modification. We also propose to investigate in detail the order in which a given E3 enzyme, particularly the anaphase promoting complex (APC), ubiquitinates its substrates. Ordering is a product of the complex kinetics of protein modification, including multistep polyubiquitination, deubiquitination, binding to carrier proteins, and proteasomal degradation. We are taking two complementary approaches to studying and modeling this network of reactions. The first is to develop simplified systems in which ubiquitination can only occur at a single site, or a few defined sites, and the kinetics of each reaction can therefore be fully understood. The second is to biochemically reconstitute the complete system, allowing us to ascertain the role of the proteasome in affecting the kinetics of ubiquitination and the ordering of substrate degradation and identify any new components that may previously have been missed. These two approaches will be connected via a mathematical model. Finally, we propose to study whether tight control of protein levels via active transcriptional, translational, and proteolytic mechanisms is frequent, or rare. Whether protein levels are controlled or variable has important implications for understanding disease susceptibility and drug efficacy, as well as for evolution PUBLIC HEALTH RELEVANCE: Protein degradation is one of the most highly regulated processes in cells and is involved in cell proliferation and cancer, inflammation, and neurodegenerative disease. As such, it is a promising target for pharmacologic intervention. This proposal studies which proteins are signaled out for regulated degradation and on a molecular level how the particular choices are made.